• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A vaccine preparation for the treatment of a disease for which a canine pathogen which can be expressed in a host canine cell comprises at least two vaccine combiners each comprising a plasmid containing a gene, wherein the combiner is a dog distemper and canine parvovirus binder. The plasmid contains one or more genes per linker, which gene is selected from the group consisting of HA for dog distemper virus and gene VP2 for F dog parvovirus.
    公开号:KR20000067866A
    申请号:KR1019997000236
    申请日:1997-07-15
    公开日:2000-11-25
    发明作者:오도네쟝-크리스또프；부샤르동안나벨；리비에르미셸
    申请人:데스꼴롱그 티에리；메리알(쏘시에떼 빠르 악씨옹 쌩플리휘에)；
    IPC主号:
    专利说明:

    POLYNUCLEOTIDE VACCINE FORMULA FOR TREATING DOG DISEASES PARTICULARLY RESPIRATORY AND DIGESTIVE DISEASES}
    The present invention relates to a vaccine formulation capable of vaccinating dogs against a number of infectious pathologies, in particular respiratory and digestive lesions. The invention also relates to a corresponding vaccination method.
    Infectious dog lesions are extremely diverse and are often difficult to control depending on the environment encountered outdoors.
    Numerous vaccines already exist, in particular Carre's disease (CDV virus), parvovirus disease (CPV virus), coronavirus disease (CCV virus), kennel cough or respiratory syndrome (PI2 virus) and rabies (Labdovirus) There is a vaccine for). Such vaccines are, more generally, live vaccines consisting of attenuated strains. This is especially the case for Carre's disease vaccine, canine adenovirus vaccine, parvovirus vaccine and canine coronavirus vaccine.
    In some cases, inactivated vaccines are also proposed as vaccines for rabies and cornavirus.
    These various vaccines are marketed individually, ie in the form of monovalent vaccines, or in the form of associated, ie multivalent vaccines.
    The multivalent associations developed so far have always had problems of compatibility and stability between the valences. In view of the different antigens used or in terms of the preparations themselves, in particular in the case of inactivation of vaccines and live vaccines, it is in fact necessary to ensure compatibility between the different potents of the vaccines simultaneously. It also addresses the problem of preservation of these bound vaccines and also their stability, especially in the presence of adjuvant. Such vaccines are typically quite expensive.
    The degree of protection and the duration of such protection may, moreover, be quite variable and also sensitive to outdoor environments. This is particularly true for vaccination of dogs, where the mosquito's antibodies prevent immunization with inactivated vaccines and even live vaccines.
    Therefore, it would be desirable to perform canine, especially dog vaccinations, with economic constraints on the use of expensive or complex vaccines.
    Vaccination testing for Carre's disease using purified formulations of the F fusion antigen and H haemagglutinin equivalent in complete Freund's adjuvant, has shown that the F antigen is a CDV virus for subunit vaccines (E.Norrby et al. , J. of Virol. May 1986: 536-541), which may constitute an interesting immunogen for protection.
    Meanwhile, in another document (P. de Vries et al., J. gen. Virol. 1988, 69: 2071-2083), CDV F and HA proteins may be advantageous in vaccinations according to the techniques of immunostimulatory complexes (ISCOMS). This is suggested.
    Mice immunized with recombinant vaccines expressing genes for the CDV F protein are protected against challenge by these viruses.
    However, this is a laboratory result, which is particularly difficult to judge under outdoor conditions.
    For parvovirosis, testing of subunit vaccines containing the main capsid protein VP2 derived from CPV virus obtained by genetic recombination from baculovirus makes it possible to show protection of immunized dogs against challenge with CPV virus. do.
    For canine herpesvirus CHV, the use of glycoproteins as a component of subunit vaccines has been studied. This study shows the induction of cross-reactions with other herpesviruses, such as FHV, but no conclusion has been drawn regarding the potential for the production of protective vaccines.
    In Lyme disease, the associated OspA and OspB induce protection in mice and dogs, and only OspA induces protection in mice, hamsters and dogs.
    Patent applications WO-A-90 11092, WO-A-93 19183, WO-A-94 21797 and WO-A-95 20660 used the technology of recently developed polynucleotide vaccines. In such vaccines it is known to use plasmids capable of expressing antigens inserted in the plasmids in host cells. All routes of administration (intraperitoneal, intravenous, intramuscular, transdermal, intradermal, mucosal, etc.) have been proposed. In addition, various means of vaccination, such as by attaching and projecting DNA onto the surface of gold particles to permeate animal skin (Tang et al., Nature 356, 152-154, 1992) and skin, muscle, fatty and breast tissue Liquid jet projectors (Furth et al., Analytical Biochemistry, 205, 365-368, 1992) which make it possible to transfect can be used.
    Polynucleotide vaccines may use, for example, naked DNA and formulated DNA in liposomes or cationic lipids.
    The prior art, on the other hand, does not provide protection in dogs by the polynucleotide method of vaccination against the disease. There is no known cause of canine coronavirus CCV and respiratory syndrome.
    In rabies, the protection of mice against toxic challenge is treated with a polynucleotide vaccine that expresses the gene for the G protein under the control of the SV40 virus early promoter (Xiang et al., Virology 199, 1994: 132-140). Demonstrated and similar results are achieved with the use of the CMV IE promoter.
    The present invention provides multivalent vaccine formulations that enable the vaccination of dogs for many pathogenic substances.
    It is still another object of the present invention to provide a vaccine formulation in which different binding values are combined while showing all the required criteria for the stability and mutual compatibility of the binding values.
    Yet another object of the present invention is to provide a vaccine formulation that is easy to use and inexpensive.
    Another object of the present invention is to provide a vaccination method with good stability which makes it possible to significantly increase the efficacy of the vaccine according to the invention or to substantially reduce the amount of vaccine required.
    Therefore, the subject of the present invention is directed to expression of the following plasmid in vivo in canine cells, in which at least two dog pathogen linkers, namely, two or more plasmids each containing an integrated plasmid having a canine virus CDV linker and a canine parvovirus CPV linker A vaccine formulation for canine pathogens having a vaccine binding value, wherein the plasmid contains, for each binding value, at least one of a gene selected from the group consisting of HA and F for the Carre disease virus and a VP2 gene for the dog parvovirus. do.
    Preferably, in the case of Carre disease linker, the plasmid (s) contain HA and F genes inserted into the same plasmid or different plasmids.
    The multivalent vaccine according to the present invention may also contain a dog coronavirus CCV linker and contain at least one gene selected from the group of S and M genes and preferably at least one plasmid containing the S gene or S and M genes. . Here too, the genes can be integrated together into the same plasmid in situations where they are inserted into or allowing their expression. The bivalent or trivalent vaccines according to the invention described above may also contain an effective binding value for the prevention of respiratory syndrome, ie a PI2 binding value containing one or more plasmids containing at least one of the HA and F genes. Preferably, the use of two HA and F genes is envisioned.
    Therefore, another advantageous linker in the case of the present invention may be associated with one or more linkers selected from the group consisting of herpesvirus CHV, Lyme disease and rabies, the plasmid being CHV for each At least one gene selected from the group consisting of the gB and gD genes for viruses, the OspA, OspB and p100 genes for B. burgdorferi (lime disease) and the G gene for rabies.
    Preferably, for herpesvirosis, two gB and gD genes are associated in two separate plasmids or a single plasmid. In Lyme disease, the OspA gene is preferred.
    Preferably, the vaccine according to the present invention, including Carre's disease and Parvovirosis linkers, will contain, as another linker, a coronavirus linker, or less preferably, a respiratory syndrome linker, or both linkers, Of course, any combination including one, some or all of coronavirus, respiratory syndrome, herpesvirus, Lyme disease and rabies combiners can be associated with both Carre disease and Parvovirosis combiners.
    A linker in the present invention means one or more antigens that provide protection from the virus of the pathogen under consideration, wherein the linker, as a subvalency, is one or more modified or natural genes from one or more strains of the pathogen under consideration. It is possible to contain.
    Pathogenic genes refer to complete genes and various nucleotide sequences, including fragments that retain the ability to induce protective responses. The concept of a gene includes a nucleotide sequence equivalent to the nucleotide sequence described above in the Examples, ie, a sequence encoding a different but identical protein. It also includes the nucleotide sequences of other strains of the pathogen under consideration, which provide protection or crossprotection specific to the strain or group of strains. It also includes nucleotide sequences that are denatured to facilitate expression in vivo by the host animal but encode the same protein.
    Different binding values are contained in the vaccine formulations according to the invention in therapeutically effective amounts.
    Preferably, the vaccine preparations according to the invention may be provided in excipients suitable for administration in an optimal dose of 0.1 to 5 ml, preferably 0.5 to 2 ml, or preferably by intramuscular route.
    The dosage will typically be 10 ng to 1 mg, preferably 100 ng to 500 μg, preferably 1 μg to 250 μg per plasmid type.
    Typically, it would be desirable to use naked plasmids simply added to vaccinated excipients such as saline (0.9% NaCl), ultrapure water, TE buffer, and the like.
    Each plasmid contains the promoter capable of ensuring expression of the gene inserted into the host cell under the control of the promoter. This will typically be a potent eukaryotic promoter and in particular a cytomegalovirus early CMV-IE promoter of human or murine origin, or optionally other origins such as rats, pigs and guinea pigs.
    More typically, the promoter may be of viral origin or cellular origin. As viral promoters other than CMV-IE, mention may be made of the SV 40 virus early or late promoter or the Rous sarcoma virus LTR promoter. It may also be a promoter of the virus from which the gene is derived, such as the promoter of the gene itself.
    Cellular promoters include promoters of the cytoskeleton gene, such as desmin promoter (Bolmont et al., Journal of Submicroscopic Cytology and Pathology, 1990, 22, 117-122; and Zhenlin et al., Gene, 1989, 78, 243 -254 or alternatively it may refer to the actin promoter.
    If several genes are present in the same plasmid, they may be present in the same transcription unit or in two different units.
    Combinations of different vaccine linkers according to the invention can be achieved by mixing polynucleotide plasmids which preferably express the antigen (s) of each linker, but it can also be envisaged to express the antigens of different linkers into the same plasmid. have.
    Also subject of the present invention is a method of vaccination of a dog consisting of administering an effective amount of a vaccine formulation as described above. This method of vaccination consists of administering the vaccine formulation in one or more doses, which dosages can be administered continuously over short periods of time and / or at long intervals.
    Vaccines according to the invention can be administered in the context of this method of vaccination, in the case of polynucleotide vaccination by different routes of administration and known administration techniques as proposed in the prior art, but the preferred route is intramuscular route. .
    The efficiency of presentation of antigen to the immune system varies from tissue to tissue. In particular, the mucous membrane of the respiratory system associates with lymphoid tissue that acts as a barrier to the invasion of pathogens and supports local immunity. Administration of the vaccine by contact with mucous membranes, in particular oral mucosa, pharyngeal mucosa and bronchial mucosa, is clearly important for vaccination of respiratory and digestive lesions.
    Thus, the mucosal route of administration forms part of the mode of administration of the invention, in particular by spraying or spraying or drinking water. To this end, it will be possible to apply the vaccine preparation and the vaccination method according to the invention.
    The subject of the present invention is also a monovalent vaccine formulation containing one or more plasmids encoding one or more genes derived from one of said viruses, said gene being the gene described above. In addition to their monovalent characteristics, such agents may have the characteristics described above in terms of gene selection, combinations thereof, composition of plasmids, volume dose, dosage, and the like.
    Monovalent vaccine formulations may be used for (i) preparation of multivalent vaccine formulations as described above, (ii) individual counterparts to actual lesions, and (iii) another type (live or inactive whole, recombinant, sub Unit) or (iv) as a booster for a vaccine as described below.
    The subject matter of the present invention is in fact also selected from the group consisting of conventional primary vaccine (single or multivalent) types of the prior art, in particular live whole vaccines, inactivated whole vaccines, subunit vaccines, recombinant vaccines. The use of one or more plasmids according to the invention for the production of a dog vaccine for vaccination of an animal primarily vaccinated with the vaccine, which primary vaccine provides (ie can contain or can express) an antigen that provides cross protection ( (S) or antigen (s) encoded by the plasmid (s).
    Remarkably, polynucleotide vaccines have a strong booster effect that results in amplification of the immune response and acquisition of long-lasting immunity.
    Typically, the primary vaccination vaccine can be selected from commercial vaccines available from various livestock vaccine procedures.
    The subject of the present invention is also a booster having a vaccination method according to the present invention and a vaccination method for primary vaccination as described above.
    In a preferred embodiment of the method according to the invention, the first vaccination is carried out by administering to the animal an effective amount of a conventional, especially inactivated, live, supervised or recombinant type of vaccine or alternatively a subunit vaccine. And, preferably, 2 to 6 weeks later, a multivalent or monovalent vaccine according to the present invention.
    A subject of the invention is also an immunization kit incorporating the primary immunization vaccine as described above and the vaccine formulation according to the invention for boosters. It also relates to a vaccine formulation according to the invention, accompanied by a leaflet indicating the use of said formulation as a booster for primary vaccination as described above.
    The present invention also relates to a process for the preparation of the vaccine formulation, ie the binders and mixtures thereof, as is apparent from this description.
    The invention will be explained in more detail by way of an embodiment of the invention with reference to the accompanying drawings.
    List of drawings
    Figure 1: Plasmid pVR1012
    Figure 2: Plasmid pAB044
    Figure 3: Plasmid pAB036
    4: Plasmid pAB024
    5: Plasmid pAB021
    Figure 6: Plasmid pAB022
    7: Plasmid pAB037
    8: Plasmid pAB038
    9: Plasmid pAB017
    10: Plasmid pAB041
    Sequence Listing
    SEQ ID NO: 1 oligonucleotide AB017
    SEQ ID NO: 2 oligonucleotide AB018
    SEQ ID NO: 3 Oligonucleotide AB085
    SEQ ID NO: 4 oligonucleotide AB086
    SEQ ID NO: 5 oligonucleotide AB053
    SEQ ID NO: 6 oligonucleotide AB054
    SEQ ID NO: 7 oligonucleotide AB045
    SEQ ID NO: 8 oligonucleotide AB048
    SEQ ID NO: 9 Oligonucleotide AB049
    SEQ ID NO: 10 Oligonucleotide AB050
    SEQ ID NO: 11: Oligonucleotide AB087
    SEQ ID NO: 12 Oligonucleotide AB088
    SEQ ID NO: 13 Oligonucleotide AB089
    SEQ ID NO: 14 Oligonucleotide AB090
    SEQ ID NO: 15 Oligonucleotide AB038
    SEQ ID NO: 16 oligonucleotide AB039
    SEQ ID NO: 17 Oligonucleotide AB011
    SEQ ID NO: 18 Oligonucleotide AB012
    Example
    Example 1 Culture of Viruses
    The virus is cultured in a suitable cell line until a cytopathic effect is obtained. Cell lines that can be used for each virus are well known to those skilled in the art. Briefly, cells sensitive to the virus used, incubated in Eagle's Minimum Essential Medium (MEM medium) or other suitable medium, are inoculated with the virus strain studied using a multiplicity of infection. Infected cells are then incubated at 37 ° C. for the period of time necessary for complete cell degeneration (average 36 hours).
    Example 2 Cultivation of Bacteria
    Borellia burgdorferi strains are cultured according to appropriate media and conditions well known to those skilled in the art. These conditions and media are particularly a. Barber (J. Biol. Med. 1984, 57, 71-75). The extraction of bacterial DNA is W. Implement according to the conditions described by Simpson et al. (Infect. Immun. 1990, 58, 847-853). Conventional techniques described by J. Sam Brock et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) can also be used.
    Example 3: Extraction of Viral Genomic DNA
    After incubation, the supernatant and lysed cells are collected and the whole virus suspension is centrifuged at 1000 g for 10 min at + 4 ° C. to remove cell debris. Virus particles are then collected by ultracentrifugation at 400,000 g for 1 hour at +4 ° C. The pellet is placed in a minimum volume of buffer (10 mM Tris, 1 mM EDTA). This concentrated virus suspension is treated with proteinase K (100 μg / ml final) for 2 hours at 37 ° C. in the presence of sodium dodecyl sulfate (SDS) (0.5% final). Viral DNA is then extracted with phenol / chloroform mixture and precipitated with 2 volumes of absolute ethanol. After standing overnight at -20 ° C, DNA is centrifuged at 10,000g for 15 minutes at + 4 ° C. The DNA pellet is dried and then placed in a minimal volume of sterile ultrapure water. It can then be digested with restriction enzymes.
    Example 4 Isolation of Viral Genomic RNA
    RNA viruses are purified according to techniques known to those skilled in the art. The genomic viral RNA of each virus is then described in P. Isolation using the "guanidium thiocyanate / phenol-chloroform" extraction technique described in Chomczynski and N. Sacchi (Anal. Biochem., 1987, 162, 156-159).
    Example 5 Molecular Biology Technology
    All structures of the plasmids are described in J. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989), using standard molecular biology techniques. Geneclean "kit (BIO 101 Inc., LA Jolla, CA).
    Example 6 RT-PCR Technology
    Specific oligonucleotides (with restriction enzyme sites at the 5 'terminus that facilitate cloning of amplified fragments) are synthesized to completely cover the coding region of the gene to be amplified (see Special Example). Reverse transcription (RT) reactions and polymerase chain reactions (PCR) are performed according to standard techniques (Sambrook J. et al., 1989). Each RT-PCR reaction is run using a pair of specific amplifiers, and as a template, the extracted viral genomic RNA is taken. The amplified complementary DNA is extracted before digestion with restriction enzymes with phenol / chloroform / isoamyl alcohol (25: 24: 1).
    Example 7: Plasmid pVR1012
    Plasmid pVR1012 (FIG. 1) is obtained from Vical Inc. (San Diego, CA, USA). Its structure is described in J. Hartikka et al. (Human Gene Therapy, 1996, 7, 1205-1217).
    Example 8 Construction of Plasmid pAB044 (CDV HA Gene)
    RT-PCR reaction according to the technique of Example 6 was carried out according to the technique of Example 4, having the following oligonucleotides: Carrene virus (CDV) (onderstepoort strain) genomic RNA (M. Sidhu et al., Virology, 1993, 193, 66-72) to isolate genes encoding CDV HA glycoproteins in the form of PstI-BamHI fragments. After purification, the 1835 bp RT-PCR product was digested with PstI and BamHI to yield a 1817 bp PstI-BamHI fragment. This fragment was ligated with vector pVR1012 (Example 7) previously digested with PstI and BamHI to give plasmid pAB044 (6676 bp) (FIG. 2):
    AB017 (35mer) (SEQ ID NO: 1):
    AB018 (37mer) (SEQ ID NO: 2):
    Example 9 Construction of Plasmid pAB036 (CDV F Gene)
    RT-PCR reaction according to the technique of Example 6 was carried out according to the technique of Example 4, having the following oligonucleotides; Carrene virus (CDV) (Onderstepper strain) genomic RNA (R. Driellen, Genbank sequence accession No. = X65509) to isolate the gene encoding the CDV F glycoprotein in the form of a NotI-BamHI fragment. After purification, the 2018 bp RT-PCR product is digested with NotI and BamHI to isolate 2000 bp NotI-BamHI. This fragment was ligated with vector pVR1012 (Example 7) previously digested with NotI and BamHI to give plasmid pAB036 (6893 bp) (FIG. 3):
    AB085 (40mer) (SEQ ID NO: 3):
    AB086 (32mer) (SEQ ID NO: 4):
    Example 10 Construction of Plasmid pAB024 (Dog Parvovirus VP2 Gene)
    The PCR reaction was carried out with canine parvovirus (CPV) (CPV-b strain) genomic DNA (C.Parrish Genbank sequence accession No. = M19296) prepared according to the technique of Example 3, having the following oligonucleotides. The gene encoding the VP2 capsid protein (CPV VP2) in the form of a BamHI fragment is isolated. After purification, the 1773 bp PCR product is digested with SalI and BamHI to isolate the 1760 bp SalI-BamHI fragment. This fragment was ligated with vector pVR1012 (Example 7) previously digested with SalI and BamHI to give plasmid pAB024 (6629 bp) (FIG. 4):
    AB053 (33mer) (SEQ ID NO: 5):
    AB054 (33mer) (SEQ ID NO: 6):
    Example 11 Construction of Plasmid pAB021 (CCV S Gene)
    The RT-PCR reaction according to the technique of Example 6 had the following oligonucleotides and was prepared according to the technique of Example 4, and canine coronavirus (CCV) genomic RNA (B. Horsburgh et al., J. Gen. Virol. 1992, 73,2849-2862) to amplify the 4374 bp fragment containing the gene encoding the CCV S glycoprotein in the form of a SalI-BamHI fragment. After purification, the RT-PCR product is digested with SalI and BamHI to isolate 4361 bp SalI-BamHI fragment. This fragment was ligated with vector pVR1012 (Example 7) previously digested with SalI and BamHI to give plasmid pAB021 (9230 bp) (FIG. 5):
    AB045 (32mer) (SEQ ID NO: 7):
    AB048 (35mer) (SEQ ID NO: 8):
    Example 12 Construction of Plasmid pAB022 (CCV M Gene)
    The RT-PCR reaction according to the technique of Example 6 was carried out according to the technique of Example 4 with the following oligonucleotides, and was prepared according to the technique of Example 4 (B. Horsburgh et al., J. Gen. Virol. 1992, 73,2849-2862) to isolate the gene encoding M glycoprotein (CCV M) in the form of a PstI-BamHI fragment. After purification, the 809 bp RT-PCR product is digested with PstI and BamHI to isolate the 792 bp PstI-BamHI fragment. This fragment was ligated with vector pVR1012 (Example 7) previously digested with PstI and BamHI to give plasmid pAB022 (5651 bp) (FIG. 6):
    AB049 (34mer) (SEQ ID NO: 9):
    AB050 (35mer) (SEQ ID NO: 10):
    Example 13: Construction of plasmid pAB037 (CHV gB gene)
    The PCR reaction was carried out in accordance with the technique of Example 3 with the following oligonucleotides, and canine herpesvirus (CHV) (Carmicre Carmichael strain) genomic DNA (K. Limbach et al., J. Gen. Virol. 1994, 75 , 2029-2039) to isolate the gene encoding the CHV virus gB glycoprotein in the form of a PstI-XbaI fragment. After purification, the 2667 bp PCR product is digested with PstI and XbaI to isolate the 2648 bp PstI-XbaI fragment. This fragment is ligated with vector pVR1012 (Example 7) previously digested with PstI-XbaI to give plasmid pAB037 (7523 bp) (FIG. 7):
    AB087 (34mer) (SEQ ID NO: 11):
    AB088 (34mer) (SEQ ID NO: 12):
    Example 14 Construction of Plasmid pAB038 (CHV gD Gene)
    The PCR reaction was carried out in accordance with the technique of Example 3 with the following oligonucleotides, and made according to the technique of Example 3 (C. Limbach et al., J. Gen. Virol. 1994, 75, genomic DNA) 2029-2039) to isolate the gene encoding the CHV virus gD glycoprotein in the form of a PstI-NotI fragment. After purification, the 1072 bp PCR product is digested with PstI and NotI to isolate 1049 PstI-NotI fragments. This fragment was ligated with vector pVR1012 (Example 7) previously digested with PstI and NotI to give plasmid pAB038 (5930 bp) (FIG. 8):
    AB089 (34mer) (SEQ ID NO: 13):
    AB090 (35mer) (SEQ ID NO: 14):
    Example 15 Construction of Plasmid pAB017 (Bornelia Bergdorferi OspA Strain)
    The PCR reaction was carried out according to the technique of Example 2, having the following oligonucleotides, and produced according to the technique of Example 2 (S. Bergstrom et al., Mol. Microbiol. 1989, 3, 479-486). ) To isolate the gene encoding the OspA membrane protein in the form of a SalI-BamHI fragment. After purification, the 842 bp PCR product is digested with SalI and BamHI to isolate 829 bp SalI-BamHI fragments. This fragment was ligated with vector pVR1012 (Example 7) previously digested with SalI and BamHI to give plasmid pAB017 (5698 bp) (FIG. 9):
    AB038 (37mer) (SEQ ID NO: 15):
    AB039 (34mer) (SEQ ID NO: 16):
    Example 16: Construction of plasmid pAB041 (rabies virus G gene)
    The RT-PCR reaction according to the technique of Example 6 had the following oligonucleotide and was prepared according to the technique of Example 4 Rabies Virus (ERA strain) genomic RNA (A. Anilionis et al., Nature, 1981, 294, 275-278) to amplify the 1589 bp fragment containing the gene encoding the rabies virus G glycoprotein. After purification, the RT-PCR product is digested with PstI and BamHI to yield 1578 bp SalI-BamHI fragment. This fragment was ligated with vector pVR1012 (Example 7) previously digested with PstI and BamHI to give plasmid pAB041 (6437 bp) (FIG. 10):
    AB011 (33mer) (SEQ ID NO: 17):
    AB012 (34mer) (SEQ ID NO: 18):
    Example 17 Purification and Preparation of Plasmids
    For the preparation of plasmids for vaccination of animals, any technique can be used which makes it possible to obtain a suspension of purified plasmids predominantly in the form of a spiral. Such techniques are known to those skilled in the art. In particular, J. Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989] alkali dissolution followed by two consecutive ultracentrifugation on a cesium chloride gradient in the presence of ethidium chloride. Technology may be mentioned. Reference may also be made to patent applications PCT WO 95/21250 and PCT WO 96/02658 which describe methods for making plasmids that can be used for immunization on an industrial scale. To prepare the vaccine (see Example 18), the purified plasmid is resuspended to obtain a solution at a high concentration (> 2 mg / ml) suitable for storage. To this end, the plasmid is resuspended in ultrapure water or TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0).
    Example 18 Preparation of Associated Vaccine
    The various plasmids required for the preparation of the associated vaccines are mixed starting with their concentrated solutions (Example 16). The mixture is prepared such that the final concentration of each plasmid corresponds to the effective amount of each plasmid. Solutions that can be used to adjust the final concentration of the vaccine can be 0.9% NaCl solution or PBS buffer.
    Specific agents, liposomes and cationic lipids can also be used in the manufacture of vaccines.
    Example 19 Vaccination of Dogs
    Dogs are vaccinated at doses of 10 μg, 50 μg or 250 μg per plasmid.
    Injection can be performed with a needle via an intramuscular route. In this case, the vaccine amount is administered in a volume of 1 or 2 ml. Injection may be performed with a needle through the intradermal route. In this case, the vaccine amount is administered at 10 points in 0.1 ml or 20 points in 0.05 ml, and the total volume is 1 ml. Intradermal injections are performed after shaving the skin (typically the flanks of the chest) or in terms of relatively hairless anatomical areas, such as on the medial side of the thigh. Liquid jet injection devices can also be used for intradermal injection.
    权利要求:
    Claims (13)
    [1" claim-type="Currently amended] To express the following plasmids in canine host cells in vivo, they contain one or more pathogen linkers, namely two or more vaccine linkers containing a plasmid incorporating genes having a Carrere virus CDV linker and a canine parvovirus CPV linker, The plasmid is a vaccine preparation for canine pathogens containing at least one of a gene selected from the group consisting of HA and F for the Carre's disease virus and a VP2 gene for canine parvovirus for each binding value.
    [2" claim-type="Currently amended] 2. The vaccine formulation of claim 1, wherein in the curry binder, the plasmid (s) contains HA and F genes inserted into the same plasmid or inserted into different plasmids.
    [3" claim-type="Currently amended] The vaccine formulation of claim 1, wherein the vaccine formulation contains a dog coronavirus CCV linker with at least one plasmid containing at least one gene selected from the group of S and M genes.
    [4" claim-type="Currently amended] The vaccine formulation according to claim 3, which contains an S gene or an S and M gene.
    [5" claim-type="Currently amended] The vaccine formulation according to any one of claims 1 to 4, further comprising an effective binding value for the prevention of respiratory syndrome, that is, a PI2 binding value containing one or more plasmids containing one or more of the HA and F genes.
    [6" claim-type="Currently amended] The vaccine formulation according to claim 5, which contains two HA and F genes of the respiratory syndrome joiner.
    [7" claim-type="Currently amended] The method according to any one of claims 1 to 6, wherein the plasmid contains at least one valency selected from the group consisting of herpesvirus disease CHV, Lyme disease, and rabies, wherein the plasmid contains the gB and gD genes for CHV virus, A vaccine formulation containing one or more genes selected from the group consisting of the OspA, OspB and p100 genes for B. bergdorferi and the G gene for rabies.
    [8" claim-type="Currently amended] 8. The vaccine formulation according to claim 7, wherein in herpesvirosis, two gB and gD genes are associated in two separate plasmids or a single plasmid.
    [9" claim-type="Currently amended] The vaccine formulation according to claim 8, which contains an OspA gene in Lyme disease.
    [10" claim-type="Currently amended] The vaccine formulation according to any one of claims 1 to 9, containing 10 ng to 1 mg, preferably 100 ng to 500 μg, more preferably 1 μg and 250 μg of each plasmid.
    [11" claim-type="Currently amended] Preparation of dog vaccines for vaccination of primary vaccinated animals using a primary vaccine selected from the group consisting of live whole vaccines, inactivated whole vaccines, subunit vaccines and recombinant vaccines Use of at least one plasmid according to any one of claims 1 to 10 wherein said primary vaccine has antigen (s) or antigen (s) encoded with plasmid (s) providing cross protection.
    [12" claim-type="Currently amended] The vaccine preparation according to any one of claims 1 to 10 and a dog vaccine selected from the group consisting of whole vaccine, inactivated whole vaccine, subunit vaccine, and recombinant vaccine are integrated together, and the primary vaccine is a polynucleotide vaccine. An immunization kit for administration of a dog vaccine and booster of a vaccine preparation in a primary vaccination, with the antigen encoded by the antigen or an antigen providing cross protection.
    [13" claim-type="Currently amended] 11. A leaflet according to any one of claims 1 to 10, carrying a leaflet indicating that the vaccine preparation can be used as a booster for primary vaccines selected from the group consisting of live vaccines, inactivated whole vaccines, subunit vaccines, recombinant vaccines. Wherein said primary vaccine has an antigen encoded by a polynucleotide vaccine or an antigen that provides cross protection.
    类似技术:
    公开号 | 公开日 | 专利标题
    DE69637472T2|2009-04-16|Recombinant vesiculovires and their use
    US6852705B2|2005-02-08|DNA vaccines for farm animals, in particular bovines and porcines
    KR970011149B1|1997-07-07|Recombinant avipox virus
    US6221362B1|2001-04-24|Avian polynucleotide formula
    Cantin et al.1987|Expression of herpes simplex virus 1 glycoprotein B by a recombinant vaccinia virus and protection of mice against lethal herpes simplex virus 1 infection
    US5620896A|1997-04-15|DNA vaccines against rotavirus infections
    DE69937571T2|2008-09-04|Mutaten cholera holotoxin as aid
    DK175500B1|2004-11-15|Composition for the protection of an animal or human from an infection caused by a respiratory syncytial virus and subunit vaccine formulation
    ES2700243T3|2019-02-14|Non-pathogenic recombinant Marek's disease virus constructs encoding infectious laryngotracheitis virus and Newcastle disease virus antigens
    Dunham2002|The application of nucleic acid vaccines in veterinary medicine
    Wallace et al.2006|Protective immune responses induced by different recombinant vaccine regimes to Rift Valley fever
    US5866553A|1999-02-02|Polynucleotide vaccine for papillomavirus
    CA2321903C|2011-05-31|Live recombinant vaccines comprising an adjuvant compound and a viral vector comprising a gene of an animal herpes or influenza virus
    KR101226869B1|2013-01-25|Multivalent canine vaccines against leptospira bratislava and other pathogens
    CA2258735C|2011-06-21|Canine herpesvirus based recombinant live vaccine, in particular against canine distemper, rabies or the parainfluenza 2 virus
    Pal et al.1999|Vaccination of mice with DNA plasmids coding for the Chlamydia trachomatis major outer membrane protein elicits an immune response but fails to protect against a genital challenge
    US6803361B2|2004-10-12|Bovine polynucleotide vaccine for the intradermal route
    Osorio et al.1999|Immunization of dogs and cats with a DNA vaccine against rabies virus
    Green et al.2001|C3d enhancement of neutralizing antibodies to measles hemagglutinin
    EP0870046B1|2009-01-07|Recombinant live vaccine containing feline herpes virus type 1, particularly for treating feline infectious peritonitis
    US8080642B2|2011-12-20|Severe acute respiratory syndrome DNA compositions and methods of use
    JP6505593B2|2019-04-24|Multivalent recombinant avian herpesvirus and vaccine for immunizing birds
    CN1329513C|2007-08-01|Avian polynucleotide vaccine formula for anti pig's diseases
    Zheng et al.2006|Construction and immunogenicity of a recombinant fowlpox virus containing the capsid and 3C protease coding regions of foot-and-mouth disease virus
    Kit et al.1991|Bovine herpesvirus-1 |-based viral vector which expresses foot-and-mouth disease epitopes
    同族专利:
    公开号 | 公开日
    AU733563B2|2001-05-17|
    WO1998003199A1|1998-01-29|
    NZ333780A|2000-10-27|
    ZA9706284B|1999-01-19|
    EP0954332B2|2014-12-31|
    KR100620302B1|2006-09-06|
    US20010009959A1|2001-07-26|
    BR9710509A|1999-08-17|
    FR2751227B1|1998-11-27|
    JP2000515521A|2000-11-21|
    DE69731309D1|2004-11-25|
    CZ300385B6|2009-05-06|
    DE69731309T3|2015-06-03|
    DE69731309T2|2005-11-17|
    KR20050087885A|2005-08-31|
    PL190150B1|2005-11-30|
    RU2002132833A|2005-01-20|
    CZ15899A3|1999-05-12|
    RU2319504C2|2008-03-20|
    EP0954332B1|2004-10-20|
    KR20060013432A|2006-02-09|
    CA2260273C|2010-12-21|
    AR034997A1|2004-04-14|
    FR2751227A1|1998-01-23|
    US6228846B1|2001-05-08|
    US6586412B2|2003-07-01|
    TW587942B|2004-05-21|
    AU3699397A|1998-02-10|
    CA2260273A1|1998-01-29|
    EP0954332A1|1999-11-10|
    PL331246A1|1999-07-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1996-07-16|Priority to FR96/09401
    1996-07-19|Priority to FR9609401A
    1997-07-15|Application filed by 데스꼴롱그 티에리, 메리알(쏘시에떼 빠르 악씨옹 쌩플리휘에)
    2000-11-25|Publication of KR20000067866A
    2005-07-20|First worldwide family litigation filed
    优先权:
    申请号 | 申请日 | 专利标题
    FR96/09401|1996-07-16|
    FR9609401A|FR2751227B1|1996-07-19|1996-07-19|Polynucleotide vaccine formula against canine conditions, especially respiratory and digestive conditions|
    [返回顶部]